The inhibitory effect of gadolinium-conjugated diflunisal on platelet function through regulating outside-in signaling of αIIbβ3 integrin

The gadolinium-conjugated diflunisal derivative ETN102 has previously been shown to suppress cyclooxygenase-2 (COX-2) levels, reduce glial activation, and attenuate neuronal damage associated with stroke. In the present study, we investigated whether ETN102 modulates platelet aggregation and activation. ETN102 selectively inhibited thrombin-induced platelet aggregation while sparing platelet responses to other agonists, including collagen, adenosine diphosphate (ADP), and U46619. ETN102 also reduced adenosine triphosphate (ATP) secretion under aggregating conditions without altering thromboxane B 2 (TXB 2 ) generation, suggesting that its antiplatelet effect is independent of the COX-1-dependent thromboxane signaling. Flow cytometric and biochemical analyses further demonstrated that ETN102 suppressed thrombin-induced αIIbβ3 integrin activation, platelet spreading, clot retraction, and the phosphorylation of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), without affecting granule secretion, intracellular Ca 2+ mobilization, or phosphorylation of Syk, PI3K, and AKT signaling molecules. In vivo , ETN102 significantly prolonged carotid artery occlusion time in a ferric chloride (FeCl 3 )-induced thrombosis model and increased tail bleeding time and hemoglobin content. In addition, ETN102 reduced infarct volume and improved neuronal survival markers in a murine model of cerebral ischemia/reperfusion injury. These findings demonstrate that ETN102 is a thrombin-context selective modulator of platelet integrin αIIbβ3 outside-in signaling, with antithrombotic and neuroprotective potential.